This invention relates generally to nucleotide compounds useful as substrates for polymerase enzymes and polynucleotides derived therefrom. More specifically, this invention relates to propargylethoxyamino nucleotides and their use in preparing fluorescently-labeled nucleotides useful as substrates for thermostable polymerases, especially their use in preparing fluorescently-labeled nucleotides as chain-terminating substrates in a fluorescence-based DNA sequencing method.
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DNA sequencing has become a vitally important technique in modern biology and biotechnology, providing information relevant to fields ranging from basic biological research to drug discovery to clinical medicine. Because of the large volume of DNA sequence data to be collected, automated techniques have been developed to increase the throughput and decrease the cost of DNA sequencing methods (Smith; Connell; Trainor).
A preferred automated DNA sequencing method is based on the enzymatic replication technique developed by Sanger (Sanger). In Sanger""s technique, the DNA sequence of a single-stranded template DNA is determined using a DNA polymerase to synthesize a set of polynucleotide fragments wherein the fragments (i) have a sequence complementary to the template sequence, (ii) vary in length by a single nucleotide, and (iii) have a 5xe2x80x2-end terminating in a known nucleotide, e.g., A, C, G, or T. In the method, an oligonucleotide primer is annealed to a 3xe2x80x2-end of a template DNA to be sequenced, the 3xe2x80x2-end of the primer serving as the initiation site for polymerase-mediated polymerization of a complementary polynucleotide fragment. The enzymatic polymerization step is carried out by combining the template-primer hybrid with the four natural deoxynucleotides (xe2x80x9cdNTPsxe2x80x9d), a DNA polymerase enzyme, and a 2xe2x80x2,3xe2x80x2-dideoxynucleotide triphosphate (xe2x80x9cddNTPxe2x80x9d) xe2x80x9cterminatorxe2x80x9d. The incorporation of the terminator forms a fragment which lacks a hydroxy group at the 3xe2x80x2-terminus and thus can not be further extended, i.e., the fragment is xe2x80x9cterminatedxe2x80x9d. The competition between the ddNTP and its corresponding dNTP for incorporation results in a distribution of different-sized fragments, each fragment terminating with the particular terminator used in the reaction. To determine the complete DNA sequence of the template, four parallel reactions are run, each reaction using a different ddNTP terminator. To determine the size distribution of the fragments, the fragments are separated by electrophoresis such that fragments differing in size by a single nucleotide are resolved.
In a modern variant of the classical Sanger technique, the nucleotide terminators are labeled with fluorescent dyes (Prober; Hobbs), and a thermostable DNA polymerase enzyme is used (Murray). Several advantages are realized by utilizing dye-labeled terminators: (i) problems associated with the storage, use and disposal of radioactive isotopes are eliminated; (ii) the requirement to synthesize dye-labeled primers is eliminated; and, (iii) when using a different dye label for each A,G,C, or T nucleotide, all four reactions can be performed simultaneously in a single tube. Using a thermostable polymerase enzyme (i) permits the polymerization reaction to be run at elevated temperature thereby disrupting any secondary structure of the template resulting in less sequence-dependent artifacts, and (ii) permits the sequencing reaction to be thermocycled, thereby serving to linearly amplify the amount of extension product produced, thus reducing the amount of DNA template required to obtain a sequence.
While these modern variants on Sanger sequencing methods have proven effective, several problems remain with respect to optimizing their performance and economy. One problem encountered when using dye-labeled terminators in combination with thermostable polymerase enzymes, particularly in the case of fluorescein-type dye labels, is that a large excess of dye-labeled terminator over the unlabeled dNTPs is required, up to a ratio of 50:1. This large excess of labeled terminator makes it necessary to purify the sequencing reaction products prior to performing the electrophoretic separation step. This clean-up step is required in order to avoid interference caused by the comigration of unincorporated labeled terminator species and bona fide sequencing fragments. A typical clean-up method includes an ethanol precipitation or a chromatographic separation (ABI PRISM(trademark) Dye Terminator Cycle Sequencing Core Kit Protocol). Such a clean-up step greatly complicates the task of developing totally automated sequencing systems wherein the sequencing reaction products are transferred directly into an electrophoretic separation process. A second problem encountered when using presently available dye-labeled terminators in combination with a thermostable polymerase is that an uneven distribution of peak heights is obtained in Sanger-type DNA sequencing.
The present invention is directed towards our discovery of a novel class of propargylethoxyamino nucleotides useful as chain-terminating dideoxynucleotides, and, as chain-extending deoxynucleotides, in a primer extension reaction, e.g., in a Sanger-type DNA sequencing or in a PCR reaction.
It is an object of the invention to provide a nucleotide which can be used to form a labeled chain-terminating nucleotide.
It is a further object of the invention to provide a chain-terminating nucleotide which includes a label.
It is yet an additional object of the invention to provide a chain-terminating nucleotide which includes a fluorescent label wherein a reduced excess concentration of such labeled chain-terminating nucleotide over an unlabeled chain-terminating nucleotide is required in a Sanger-type DNA sequencing process.
It is another object of the invention to provide a labeled chain-terminating nucleotide which results in a more even distribution of peak heights in a Sanger-type DNA sequencing process.
It is an object of the invention to provide a nucleotide which can be used to form a labeled chain-extending deoxynucleotide.
It is a further object of the invention to provide a chain-extending deoxynucleotide which includes a label.
It is an additional object of the invention to provide methods including a primer extension reaction utilizing the propargylethozyamino nucleotides of the invention.
In a first aspect, the foregoing and other objects of the invention are achieved by a nucleoside compound having the structure: 
wherein the variable substituents R1-R2 and W1-W3 are defined as follows. R1 and R2 taken separately are xe2x80x94H, lower alkyl protecting group, or label. In a preferred embodiment, one of R1 and R2 is label, the label preferably being a fluorescein-type dye or a rhodamine-type dye. B is a 7-deazapurine, purine, or pyrimidine nucleoside base, preferably uracil, cytosine, 7-deazaadenine, or 7-deazaguanosine. When B is purine or 7-deazapurine, the sugar moiety is attached at the N9-position of the purine or deazapurine, and when B is pyrimidine, the sugar moiety is attached at the N1-position of the pyrimidine. When B is a purine, the adjacent triple-bonded carbon is attached to the 8-position of the purine, when B is 7-deazapurine, the adjacent triple-bonded carbon is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, the adjacent triple-bonded carbon is attached to the 5-position of the pyrimidine. W1 is xe2x80x94H or xe2x80x94OH. W2 is xe2x80x94OH or a moiety which renders the nucleoside incapable of forming a phosphodiester bond at the 3xe2x80x2-position. W3 is xe2x80x94PO4, xe2x80x94P2O7, xe2x80x94P3O10, phosphate analog, or xe2x80x94OH.
In a second aspect, the invention includes a method for performing a primer extension reaction including the following steps: providing a template nucleic acid; annealing an oligonucleotide primer to a portion of the template nucleic acid; and adding primer-extension reagents to the primer-template hybrid for extending the primer. In an important aspect of the invention, the primer extension reagents include a propargylethoxyamino nucleoside compound having the structure described above.